WO2007034509A2 - Formulation d'un facteur de stimulation de colonies de granulocytes de recombinaison et procede de preparation correspondant - Google Patents

Formulation d'un facteur de stimulation de colonies de granulocytes de recombinaison et procede de preparation correspondant Download PDF

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Publication number
WO2007034509A2
WO2007034509A2 PCT/IN2006/000371 IN2006000371W WO2007034509A2 WO 2007034509 A2 WO2007034509 A2 WO 2007034509A2 IN 2006000371 W IN2006000371 W IN 2006000371W WO 2007034509 A2 WO2007034509 A2 WO 2007034509A2
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range
csf
formulation
protein
buffer
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PCT/IN2006/000371
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English (en)
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WO2007034509A3 (fr
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Uma Devi Komath
Jayaram Chigurupati
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Zenotech Laboratories Limited
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Publication of WO2007034509A3 publication Critical patent/WO2007034509A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

Definitions

  • the present invention provides a stable formulation for recombinant human Granulocyte - Colony Stimulating Factor (G-CSF) in the pH range of 4.0 to 5.0 and a conductivity value greater than 1.0 milliSiemens/cm, comprising of L-methionine, to suppress the formation of oxidized G-CSF during storage.
  • G-CSF Granulocyte - Colony Stimulating Factor
  • This invention also includes the process for preparing stable G- CSF formulation
  • G-CSF Granulocyte - colony stimulating factor
  • E. coli produced G-CSF is a 175 amino acid polypeptide chain containing an extra methionine at its N- terminus. This protein has been produced by expressing a G-CSF gene in E. coli and purifying the protein product to homogeneity.
  • oxidized forms of the protein that contain oxidized methionine at position 122 can be separated from the forms containing oxidized methionines at positions 127 or 138 and the native protein by regular reverse phase HPLC separation procedures.
  • G-CSF Several pharmaceutical preparations of G-CSF have been described in earlier patents that are suitable for administration to humans.
  • the emphasis in various formulation strategies is on preventing the formation of higher order aggregates of the protein by disulphide exchange involving the free cysteine residue.
  • the free cysteine residue the occurrence of methionine residues that can undergo oxidation in the presence of free radicals in solution, also contribute to the formation of altered conformational forms of G-CSF that can initiate the formation of higher order aggregates of the protein in solution.
  • a more effective formulation for G-CSF would be the one that includes L- solution.
  • G-CSF G-CSF sequence formulated in a buffer of suitable pH and conductivity value that maintains the structural integrity of the molecule.
  • Uwe Michaelis (Boehringer Mannheim) describes an aqueous pharmaceutical preparation of G-CSF comprising of a therapeutic amount of the protein in a buffer selected from the group of citrate, maleate, phosphate and arginine or their salts thereof and a surfactant at a pH value between 7.0 and 8.0.
  • This formulation is described as one with a long shelf life, which can tolerate mechanical stress and has a physiologically compatible pH value.
  • a stable formulation of G-CSF contains besides the active ingredient, at least one substance selected from a pharmaceutically acceptable surfactant, saccharide, protein and a high molecular weight compound.
  • This formulation is described as being highly stable and capable of preventing the loss of active component from solution by adsorption to container surfaces, association, polymerization or oxidation of the said component.
  • the patent assigned to Rhone-Poulenc Rorer S. A. in US 5554150 methods and devices are described for the delivery of G-CSF to the patients.
  • the method comprises continuous sub-cutaneous delivery of a solution comprising granulocyte colony stimulating factor to the patient.
  • Devices for continuous administration are disclosed as well as the preferred composition of the solution which is also described in patent GB 2163631.
  • the solution consists of G-CSF, serum albumin, non-ionic surface active agent, saccharide, disodium phosphate, monosodium phosphate, sodium chloride and water.
  • the said formulation has an effective amount of hydrophobic protein like G-CSF or IL-2 at acidic pH and advantageously a low conductivity which is less than 1000. mu. mhos/cm.
  • the pH is about 2.5 to 4.0 and in a preferred embodiment, no buffer is present.
  • the method involves combining the protein advantageously with acid without the addition of a salt to make a pharmaceutically acceptable formulation.
  • it can also contain a tonicity modifier.
  • EP 1197221 describes a stable lyophilized, CHO-cell derived G-CSF formulation in the pH range of 5.0 - 7.0, more specifically around 6.5 and containing mannitol, Tween 20 and amino acids.
  • the amino acids are one or more selected from a group of lysine, histidine, arginine, aspartic acid and glutamic acid; and one or more amino acids selected from a group consisting of phenylalanine, tryptophan and leucine; and with methionine.
  • the formulation is claimed to have a residual ratio of G-CSF of 90% or more after long term storage testing and methionine - oxidized G-CSF of 1% or less.
  • G-CSF Granulocyte -Colony Stimulating Factor
  • a tonicity modifier like mannitol or sorbitol in the range of 20 to 70 mg/ml
  • a surfactant like Polysorbate20 or PolysorbateSO in the concentration range of 0.001 to 0.01%
  • an amino acid which is an oxidation suppressant for the methionine residues in the protein in the concentration range of 0.05 to 0.5mg/ml in an acidic buffer in the conductivity range greater than one milliSiemens per cm and a pH range of 4.0 to 5.0.
  • the present invention also provides a process for preparing a stable G-CSF formulation comprising the steps of formulating the: purified protein G-CSF in the concentration range of 0.2 to 0.8 mg/ml; adding a tonicity modifier in the range of 20 to 70 mg/ml; adding a surfactant in the concentration range of 0.001 to 0.01%; in an acidic buffer in the conductivity range greater than one milliSiemens per cm and a pH value in the range of 4.0 to 5.0.
  • Figure 1 is a RP-HPLC separation of recombinant human G-CSF from its oxidized forms.
  • the form of G-CSF with methionine 122 oxidised has a retention time around 26 minutes, whereas the form having methionines at 127 and 138 have a retention time of around 32 minutes on a C- 18 RP-HPLC column.
  • the intact G-CSF molecule, with no methionines oxidized, has a retention time of about 33 min on the same column.
  • the solvent system used is acetonitrile /TFA.
  • B) Without methionine addition DETAILED DESCRIPTION OF THE INVENTION
  • G-CSF Granulocyte — Colony Stimulating Factor
  • G-CSF refers to a protein that has the sequence of the naturally occurring human G-CSF or the recombinant form with or without an additional N-terminal methionine residue. It can also encompass derivatives of the molecule which are structural analogues or derivatives with amino acid additions or deletions to the naturally occurring G-CSF molecule.
  • G-CSF is formulated in a buffer of acidic pF£ comprising of a tonicity modifier, a surfactant and an amino acid, which is an oxidation suppressant for the methionine residues in the protein.
  • the buffers chosen are in the conductivity range of 1.0 to 6.0 milliSiemens, more preferably in the 1.1 to 2.0 milliSiemens range and a pH range of 4.0 to 5.0.
  • Various buffer substances can be used in the formulation like acetic acid, phosphoric acid, citric acid or lactic acid and they can be present either in the free acid form or as salts of alkali, alkaline earth metals or ammonia. The more preferred substances being acetate and phosphate.
  • the desired pH value is obtained by the addition of base or acid as required for attaining the final pH value.
  • the conductivities chosen are such that the values are suitable for therapeutic administration.
  • the concentrations of buffer solutions that can be used are in the range of 1 to 20OmM, but more preferably in the 15 to 15OmM range of concentration and most preferably in the 40 to 100 niM range.
  • the formulation also contains other excipient substances that provide an isotonicity that is close to the physiological levels in the human body.
  • the necessary isotonicity value is conferred by the use of neutral auxiliary substances like sugars and sugar alcohols. These are non-ionic and extremely well-soluble in water at high concentrations. Examples of such substances are mannitol, sorbitol, glycerol and other similar sugars and sugar alcohols.
  • an oxidation retarding agent is also added to the formulation buffer to prevent the formation of multiple oxidized forms of G-CSF.
  • the amino acid sequence of the G-CSF molecule is known to have three methionine residues, at positions 122, 127 and 138.
  • the oxidized forms of G-CSF are formed by the oxidation of one or more of these methionine residues in the protein.
  • Reverse phase HPLC methods are used to separate and identify the various methionine oxidized forms of G-CSF which arise during purification, formulation or storage. The oxidation state of a protein after formulation remains stable, if the protein is not exposed to an oxidizing environment during long term storage.
  • G-CSF and other protein formulations containing oxidants protein stability can be maintained throughout the shelf life of the product, by the addition of specific amino acids capable of protecting the protein from the damaging effects of oxidative free radicals.
  • certain lots of G-CSF have been shown to exhibit an increased tendency to produce oxidized forms. This was predominantly due to a non-ionic surfactant Polysorbate (present in the formulation buffer) having a tendency to produce peroxides, that cause oxidation of methionine residues in the protein.
  • Polysorbate a common excipient in the therapeutic formulations of G-CSF, is generally added in liquid formulations to eliminate the formation of particulates that are occasionally observed at elevated temperatures.
  • Methionine can be added as a free amino acid or as its salt containing the D, L or DL forms.
  • the L- variant is however, preferred.
  • the preferred final concentration is in the range of 0.01 to 1.0 mg/ml and more preferably in the 0.05 to 0.5 mg/ml.
  • Formulations of the present invention may contain in addition to methionine, other agents like non-ionic surfactants and sugars:
  • the non-ionic surfactant preferred in this case is from the sorbitan fatty acid ester and polyoxyethylene sorbitan fatty acid esters series, that are added in the concentration range of 0.001 to 0.01%.
  • the sugar molecule preferred belongs to the group of sugar alcohols consisting of molecules such as sorbitol and mannitol. The concentration range of the sugars is in the 5 - 100 mg/ml, the more preferred range being 20 to 70 mg/ml.
  • the active ingredient in this invention can be cytokines, more preferably Granulocyte- Colony Stimulating Factor, in its natural or recombinant form, derivatives of G-CSF or functional polypeptides of the same molecule.
  • the concentration of the active substance is in the range of 0.1 to 1.0 mg/ml more preferably in the range of 0.2 to 0.8 mg/ml.
  • Methodology for preparation of formulated G-CSF Purified G-CSF protein or its derivative is buffer exchanged with 4OmM to 10OmM concentration of the formulation buffer in the pH range of 4.0 to 5.0. To this, excipients are added in stated amounts either in solid form or from higher molarity stock solutions. Sugars or sugar alcohols like sorbitol or mannitol are preferably added in solid form at a concentration of 20 - 70 mg/ml. Surfactants are added at a concentration of 0.001 to 0.01 % either directly or from a freshly prepared stock solution in formulation buffer of the surfactant at a higher molarity.
  • the amino acid methionine is also added from a stock solution to give a final concentration in the range of 0.05 to 0.5 mg / ml.
  • the excipient substances chosen are of the formulation grade and essentially free of endotoxins.
  • the formulated protein solution is stirred gently at room temperature to ensure a thorough and homogenous mixture of the contents.
  • the final pH and conductivity values are checked and re-adjusted to the desired value, if required, by the addition of small amounts of acid or base component of the buffer.
  • the solution so obtained is filtered through a 0.22 micron or similar sterilizing grade filtration membrane and filled into suitable containers like vials or syringes for ease in therapeutic administration.
  • Methodology for stability testing of formulated G-CSF This example relates to the stability indicating tests for different formulations of G-CSF which are held at defined storage temperatures. The temperature parameters are set, such that the accelerated and real time stability of the formulated protein can be ascertained.
  • the stability determining parameters are initially defined and various biochemical and biological tests are conducted at different intervals of time to ensure that the quality of the protein falls within the defined specifications.
  • a very useful test method to determine the presence of aggregates in the G-CSF formulated sample is by denaturing polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis, when done under non-reducing conditions can effectively discern the presence of multimeric forms of the protein produced by the process of aggregation.
  • the stability of the formulation to aggregation or degradation on storage is alternately determined and quantitated by the method of size exclusion HPLC (SE-HPLC) and reverse phase HPLC (RP-HPLC) analysis.
  • SE-HPLC size exclusion HPLC
  • RP-HPLC reverse phase HPLC
  • a C- 18 RP-column is connected to the HPLC system.
  • the mobile phase used is a triflouroacetic acid / acetonitrile solvent system and the wavelength of detection is set at 280 nm.
  • formulated G-CSF solutions held at different storage conditions, are injected and the number of peaks obtained and their retention times are noted and compared with the peak area and RT of an external reference standard.
  • the SE-HPLC column is equilibrated using phosphate buffer in the pH range of 2.0 to 4.0 and preconditioned using an aggregated G-CSF solution.
  • the stability test samples are injected to indicate the presence and quantity of aggregated protein.
  • Changes in potency during storage are determined in an in vitro cell proliferation based bioassay on a G-CSF responsive cell line like NFS-60.
  • the potency of the sample is determined by comparison of the dose response curve of the test samples with that of a reference standard by parallel line analysis of data.
  • This example relates to the stability of different formulations when held at defined storage temperatures (25 ⁇ 2 °C and 5 ⁇ 3 0 C).
  • G-CSF was formulated at different concentrations of the protein, in the range of 0.2 to 0.8 mg/ml, in the pH range of 4.1 to 4.8 in buffers with conductivities in the range of l.lmilliSiemens to 2.0 milliSiemens.
  • the presence of different sugar alcohols like sorbitol or mannitol were also tested along with the addition of amino acids like methionine in the final buffers.
  • the percentage of aggregates in the sample was determined by SE-HPLC analysis.
  • a SE- HPLC column, TSK G 3000 SW XL , 5 um and 7.8 x 300 mm dimensions was run at 1.0 ml/min flow rate with 10OmM PO4 buffer of pH 2.5.
  • the protein peak was detected at 280nm wavelength and quantified using the peak area calculation with respect to a known quantity of reference standard.
  • the tables below will indicate that the percentage of aggregates in the sample is within limits at different conditions of protein concentration, buffer pH, conductivity and presence of methionine. The acceptable limit is ⁇ l% aggregates.
  • the bioactivity of G-CSF was determined by a cell proliferation assay on NFS-60 cells.
  • the activity was measured by correlating the reduction of MTT [3-(4, 5-dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium bromide], a soluble tetrazolium salt, to an insoluble formazan crystal.
  • the potency was estimated by comparison with a standard reference sample of known potency.
  • the following tables 6-10 illustrate the stability of the potency in the formulation under different conditions of protein concentration, buffer pH, conductivity and addition of methionine. The acceptable range is 22.5 MIU to 37.5 MIU.
  • a liquid pharmaceutical solution of G-CSF was prepared as described in Example 3 and the stability of the formulation was estimated by determining the extent of oxidation.
  • the formulation solutions were prepared by the addition of the amino acid methionine and the solutions without methionine were used as controls (Tables 11-13). All the solutions were stored at 25°C for accelerated stability and 2 to 8°C for real time stability studies.

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Abstract

La présente invention concerne un procédé de préparation d'une formulation thérapeutique liquide stable de G-CSF, dans laquelle la protéine est placée dans un tampon d'une conductivité supérieure à 1,0 milliSiemens/cm et dans une plage de pH comprise entre 4,0 et 5,0, en présence d'un tensioactif, d'un modificateur de pression osmotique effective et d'un acide aminé qui supprime l'oxydation du médicament protéique. On obtient ainsi de bonnes stabilités de conservation à des conductivités et des valeurs de pH supérieures sans ajouter de protéines stabilisatrices d'origine humaine ou animale.
PCT/IN2006/000371 2005-09-19 2006-09-19 Formulation d'un facteur de stimulation de colonies de granulocytes de recombinaison et procede de preparation correspondant WO2007034509A2 (fr)

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IN1317CH2005 2005-09-19
IN1317/CHE/2005 2005-09-19

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WO2007034509A2 true WO2007034509A2 (fr) 2007-03-29
WO2007034509A3 WO2007034509A3 (fr) 2011-05-12

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2399572A1 (fr) 2010-06-22 2011-12-28 Sandoz AG Stockage à long terme de G-CSF humain recombinant non glycosylé

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5451662A (en) * 1987-10-23 1995-09-19 Schering Corporation Method of purifying protein
US20040037803A1 (en) * 2000-09-01 2004-02-26 Yasushi Sato Solution preparations stabilized over long time

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5451662A (en) * 1987-10-23 1995-09-19 Schering Corporation Method of purifying protein
US20040037803A1 (en) * 2000-09-01 2004-02-26 Yasushi Sato Solution preparations stabilized over long time

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2399572A1 (fr) 2010-06-22 2011-12-28 Sandoz AG Stockage à long terme de G-CSF humain recombinant non glycosylé
WO2011161165A1 (fr) 2010-06-22 2011-12-29 Sandoz Ag Stockage de longue durée de g-csf humain recombiné non glycosylé
US8449873B2 (en) 2010-06-22 2013-05-28 Sandoz Ag Long-term storage of non-glycosylated recombinant Human G-CSF
US8784794B2 (en) 2010-06-22 2014-07-22 Sandoz Ag Long-term storage of non-glycosylated recombinant human G-CSF
US9387233B2 (en) 2010-06-22 2016-07-12 Sandoz Ag Long-term storage of non-glycosylated recombinant human G-CSF

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